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Abstract

Background

Biological oxidation of methane (CH4) is an essential ecosystem function. Accumulating evidence indicated that this function is mediated by associations of methanotrophic bacteria (MOB) with non-methanotrophic partners; together referred to as a methanotrophic interactome. Given the potency of CH4 as a greenhouse gas, a thorough understanding of how these interactomes exert an effect on methane oxidation is of special interest. Furthermore, MOB - non-MOB associations could be exploited for sustainable biotechnological applications in light of the renewed interest in MOB as natural and cost-efficient biocatalysts. The selectivity of MOB for non-MOB partners, as well as the stimulation of MOB activity (CH4 oxidation rate, MOR) with increasing non-MOB richness have both been recently described for a single batch incubation period. Therefore, we hypothesized that during repeated co-cultivation of MOB with non-MOB, ecological sorting would guide the methanotrophic interactome towards its optimal composition, which could additionally boost functionality (MOR).

Methods

Co-cultures of 8 non-MOB partners with a single alpha- or a single gammaproteobacterial MOB were repeatedly sub-cultivated. In every cycle, the headspace CH4 concentration was measured to over time to determine the MOR, while headspace CO2 concentrations and total protein in the culture were determined to track the fate of CH4-derived carbon (catabolism and assimilation respectively). Finally, the relative abundance of each co-culture partner was assessed using a 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE).

Results and Discussion

While no significant improvement of functionality was observed, the biological variability of MOR was stabilized by co-cultivation with non-MOB partners. Overall, higher biomass yields were obtained when MOB were co-cultivated with non-MOB partners and the alphaproteobacterial MOB appeared to be able to support more non-MOB biomass than the gammaproteobacterial MOB, which could be linked to the proposed life-strategies of these clades. A clear partner selection was observed as only 4 out of 8 initial partners were found to persist during repeated cycles of co-cultivation. While 2 of the persisting partners could coexist with either MOB type, the other two were more restricted to a specific MOB. Differential metabolic potential of non-MOB was resolved by genome mining publicly available genomes; our attempt to find clues for the partner selectivity did not reveal a clear link with the potential for C1-compound metabolism. However, genes for sugar metabolism (fructose, mannose, sucrose) were restricted to the persisting partners while genes encoding an ATP-dependent vitamin B12 importer were restricted to the non-persisting partners, underlining the importance of metabolic exchange in the methanotrophic interactome.

Author Comment

This is a submission to PeerJ for review.

Supplemental Information

Overview of the experimental design

The design is a split-split plot design with repeated measures. Cx (x=1-5) represents the cycle of (co)-cultivation, where n represents the total amount of headspace measurements available for each cycle.

DGGE pattern among the cycles

16S rRNA gene DGGE was performed as described in materials & methods. Using BioNumerics (Applied Maths, version 5.1) band classes were assigned. Only the most abundant band class of a pure strain loaded on the gel was selected as “representative” band class, hence correcting for possible ghost bands. Samples were color-coded according to the cycle they belonged to. R45002 represents Methylomonas methanica NCIMB 11130T. +8HET refers to co-cultivation incubations. 8HET as such are lanes from the negative control of the 8 non-MOB incubated without the MOB. Cycle “none” refers to pure cultures loaded in the lane. Color codes (Column “Code”) are depicting the (co-) incubation cycle (column “Cycle”). In the column MOB “NOMOB” refers to lanes where amplicon of the (pure) cultures of the non-MOB partners were loaded. Fuzzy clustering was performed using the Jaccard distance (aware of band intensity) and UPGMA linkage.

Methane removal profiles

CH4(t=t)/CH4(t=t0) profiles for all experimental conditions and cycles. Observation points represent average CH4(t=t)/CH4(t=t0) of either triplicate (MOB alone) or quadruplicate (MOB with heterotrophs) measurements, except for the heterotroph control incubation without methanotrophs, where only one biological replicate was used. A dashed horizontal red line represents 25% methane removal from the initial concentration. Shaded areas represent 95% confidence intervals on the observations.

Box-and-whisker plots for methane oxidation ratio's (MOR) for each cycle per treatment

MOR is expressed as mmol CH4 oxidized per liter of broth per hour. MOR is corrected for losses during incubation by means of a negative control with only heterotrophs (HET). LMG: pure culture cultivation of Methylosinus sp. LMG 26262. LMG+HET: co-cultivation of Methylosinus sp. LMG26262 with 8 non-MOB partners as described in materials & methods. R: pure culture cultivation of Methylomonas methanica NCIMB 11130T. R+HET: co-cultivation of Methylomonas methanica NCIMB 11130T with 8 non-MOB partners as described in materials & methods. Black dots in the boxplot represent the median MOR.

Comparative genomics of C1 metabolism

Microsoft Excel spreadsheet of C1 modules compiled from Chistoserdova (2011) and RAST subsystems/scenarios for the metabolic processes. “?” indicates that it is unclear if a certain function is correctly assigned or (in)complete. “Maybe” indicates that a gene was found that could be involved in the functionality though it is not clear if it is enough for the functionality or the correct gene. Individual gene names were given when appropriate.

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Frederiek - Maarten Kerckhof conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables.

Charlotte De Rudder conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Varvara Tsilia performed the experiments, reviewed drafts of the paper.

Adrian Ho conceived and designed the experiments, reviewed drafts of the paper.

Kim Heylen conceived and designed the experiments, reviewed drafts of the paper.

Nico Boon conceived and designed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.

Data Deposition

The following information was supplied regarding data availability:

The raw data has been supplied as a Supplemental Dataset.

Funding

This work was funded by the Ghent University “Geconcerteerde onderzoeksactie” on Sustainable methanotrophs (BOF09/GOA/005). K. Heylen was supported by the Fund for Scientific Research Flanders for a position as a postdoctoral fellow (FWO15/PDOH1/084). F.-M. Kerckhof was supported by the Inter-University Attraction Pole (IUAP) ‘μ-manager’ funded by the Belgian Science Policy (BELSPO, P7/25). C. De Rudder was supported by the ProCure IWT SBO project (IWT SBO 150052). A. Ho was supported by BE-Basic grant F03.001 (SURE/SUPPORT). R. Props was supported by Ghent university (BOFDOC2015000601) and the Belgian Nuclear Research Centre (SCK•CEN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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